The Regulatory Effect And Mechanism Of Estrogen On Peripheral AIRE Expression Via ERα And DNMT

Autoimmune regulator（AIRE）is one of the important transcriptional regulators to maintain immune tolerance.Abnormal expression of AIRE will lead to autoimmune diseases of multiple organs.AIRE mainly participates in thymic negative selection and maintains immune tolerance by regulating the ectopic expression of tissue restricted antigens（TRAs）in thymic medullary epithelial cells（mTECs）.The deletion or mutation of AIRE is easy to lead to autoimmune diseases of multiple organs,that is,autoimmune polyglandular syndrome type Ⅰ（APS-1）.In addition,AIRE is also expressed in peripheral lymph node stromal cells and spleen dendritic cells（DC）,and also has the role of regulating TRAs.However,it is found that the TRAs regulated by AIRE in the periphery is not exactly the same as that in the thymus.In addition,our research group found that the expression of AIRE in peripheral antigen-presenting cells can also maintain peripheral immune tolerance by regulating Treg cells.However,the molecular mechanism of regulating AIRE is not clear.In addition,most autoimmune diseases are common in women,and estrogen has a high level expressin in women.Therefore,we speculate whether estrogen is involved in the regulation of peripheral AIRE,thus affecting the susceptibility of women to autoimmune diseases?Methylation is one of the important reasons affecting gene expression.DNA methyltransferases（DNMTs）are the key factors involved in methylation.Some studies have shown that estrogen can affect the expression of DNMTs through estrogen receptor（ER）.Therefore,we speculate that if estrogen can regulate the expression of peripheral AIRE,whether it affects DNMTs through ER,eventually lead to abnormal expression of AIRE,finally destroy immune tolerance,and lead to women’s susceptibility to autoimmune diseases?In order to explore this problem,we conducted the following research:Ⅰ.Observation of estrogen on the expression and function of AIRE in lymph node stromal cells and spleen DCcells in vitro1.Effects of estrogen on AIRE expression in lymph node stromal cells and splenic DCIn order to study the effect of estrogen on the expression of AIRE in lymph node stromal cells and splenic DC,the lymph nodes in mice were isolated and obtained for the cultivation of lymph node stromal cells.At the same time,the splenic DC were isolated by magnetic bead separation.The cell purity was identified by flow cytometry,which proved that the corresponding cells had been obtained.Further,the expression differences of AIRE in the above two cells from mice with different sexes were compared by qPCR.The results showed that the expression of AIRE in the periphery of female mice was lower than that of male mice.Lymph node stromal cells and splenic DC were treated with different concentrations of estradiol and dihydrotestosterone（DHT）for 24 hours.QPCR detection showed that estradiol significantly reduced the expression of AIRE.After DHT treatment,the expression of AIRE increased significantly.Combination of estrogen and DHT at different concentrations were used to treat lymph node stromal cells and splenic DC.QPCR results showed that the simultaneous application could antagonize each other,and the inhibitory effect of estradiol was stronger.In splenic DC,we also found that estradiol significantly down regulated the expression of AIRE,and DHT only slightly up regulated the expression of AIRE,which was not statistically significant.These results suggest that estrogen can significantly down regulate the expression of AIRE in peripheral lymph node stromal cells and splenic DC,but the specific mechanism is not clear.2.Effects of estrogen on the expression of related TRA in lymph node stromal cells and splenic DCIn order to further understand whether estradiol and DHT affect the role of AIRE in lymph node stromal cells and splenic DC,we also detected the expression of TRA,hypothyroidism related antigen NALP5as known to be regulated by AIRE by qPCR.The results showed that after estradiol,the estrogen decreased the expression of NALP5 both in lymph node stromal cells and spleni DC,while DHT had the opposite effect.This is consistent with the expression change of AIRE.This suggests that estrogen may affect the related TRA by inhibiting the expression of peripheral AIRE,so as to affect its immune tolerance.3.Estrogen affects the effect of lymph node stromal cells and splenic DC cells on the differentiation of naive CD4+T cellsIn order to explore the regulatory effect of estrogen on AIRE,in addition to the corresponding TRA,it also has an effect on the differentiation of CD4+T cell subsets.We co-cultured lymph node stromal cells or splenic DC with naive CD4+T cells for 72 hours,then detected the subset of CD4+T cell by flow cytometry.It was found that after treating with estradiol and DHT,there was no significant change in the proportion of Th1,Th2,Th17,Treg and TFH cells compared with the control group.This suggests that sex hormones may play a role by regulating AIRE dependent TRA,rather than by regulating CD4+T cell subsets.Ⅱ.To explore the effect of estrogen via ERa and DNMT on the expression of peripheral AIRE in vitro1.Effects of estrogen on AIRE methylationin in lymph node stromal cells and splenic DCsIn order to observe whether estrogen can affect the methylation level of AIRE in lymph node stromal cells and splenic DC cells,we detected the methylation level of AIRE in estradiol treated lymph node stromal cells and splenic DC cells by BSP methylation sequencing method.The results showed that under the action of estradiol,the methylation levels of AIRE promoter at 24,51,and 105 in lymph node stromal cells were higher than those in the control group,and the methylation levels of AIRE promoter at 32 and 54 in spleen DC were higher.In contrast to the control group,while DHT did not upregulate AIRE methylation levels in lymph node stromal cells,DHT also did not upregulate AIRE promoter methylation levels in splenic DCs.This suggests that estrogen affects the expression of AIRE by methylating the AIRE2.Effects of estrogen on DNMT in lymph node stromal cells and splenic DCsIn order to understand whether estrogen may mediate the methylation of AIRE gene by affecting the expression of DNMT,we first detected the changes of the expression of DNMT1,DNMT3a and DNMT3b in lymph node stromal cells and splenic DC after treatment with estradiol.The results showed that the expression of DNMT1,DNMT3a and DNMT3b in lymph node stromal cells and splenic DC were significantly upregulated under the action of estrogen.And DNMT3b was also up-regulated,but there was no statistical significance.This suggests that estrogen may promote the methylation of AIRE by promoting the expression of DNMTs.3.The effect of ERa antagonism on AIRE and DNMT in lymph node stromal cells and splenic DCIn order to explore whether the above effects play a role through estrogen receptor（ER）expressed in lymph node stromal cells and splenic DC,firstly,we detected the expression of ERα and ERβ in lymph node stromal cells and splenic DC by qPCR The expression of ERα was found significantly higher than that of Erβ.It is likely that ERα is the main receptor mediating estrogen signaling.Further,we used different concentrations of ERα inhibitor ICI182780 to treat lymph node stromal cells and splenic DC.The results showed that the expression of AIRE mRNA in cells increased after inhibition.At the same time,the flowcytometry results also confirm this phenomenon.It was found that the expression of DNMTs in lymph node stromal cells decreased after blocking the estrogen receptor.These results suggest that estrogen can regulate the expression of AIRE in peripheral lymph node stromal cells and splenic DC cells through ERα,and its role may be played by affecting DNMTs.4.The effect of DNMT antagonism on AIRE expression in lymph node stromal cells and splenic DCIn order to further clarify the effect of DNMTs on the expression of AIRE in lymph node stromal cells and splenic DC,we treated lymph node stromal cells and spleen DC cells with DNMTs inhibitor（SGI-1027）.The results showed that the expression of AIRE in lymph node stromal cells increased with the increase of SGI-1027 concentration,and the expression level of AIRE dependent TRA-nalp5 mRNA increased at the same time,and the effect was the most significant at 20 μM SGI-1027.The results were the same when treated on splenic DC.This suggests that estrogen can regulate the expression of AIRE in peripheral lymph node stromal cells and splenic DC through ERα-DNMTs.Ⅲ.To explore the effect of estrogen on AIRE expression in lymph node stromal cells and splenic DC via ER and DNMT in vivo1.The effect of estrogen on AIRE of lymph node stromal cells and splenic DC was verified in vivo using an ovariectomized mouse modelIn order to further confirm the regulatory effect of estrogen on the expression of AIRE in mice,the results of qPCR and flow cytometry showed that the expression of AIRE was up-regulated in mouse peripheral lymph node stromal cells and splenic DC after oophorectomy.Two weeks after orchiectomy,the expression of AIRE in peripheral lymph node stromal cells and spleen DC cells decreased compared with the sham operation group.It is suggested that estrogen can inhibit the expression of AIRE in lymph node stromal cells and splenic DC cells,while androgen has the opposite effect.2.The effect of ERa antagonism on the expression of AIRE in lymph node stromal cells and splenic DC in model miceTo further clarify if estrogen affect the expression of AIRE in peripheral lymph node stromal cells and spleen DC cells through ERα in vivo,we injected estrogen receptor inhibitor（ICI182780）subcutaneously into mice.Compared with the control group,inhibiting the expression of estrogen receptor can up regulate the expression of AIRE in lymph node stromal cells and splenic DC.The results suggest that estrogen can down regulate the expression of AIRE through estrogen receptor in mice.3.The effect of antagonizing DNMT on the expression of AIRE in lymph node stromal cells and splenic DC in model miceIn order to further verify whether DNMTs can also regulate the expression of AIRE in vivo,we injected DNMTs inhibitor（SGI-1027）subcutaneously into mice,and detected the expression of AIRE in lymph node stromal cells and splenic DC by flow cytometry.The results showed that inhibiting the expression of DNMTs could up regulate the expression of AIRE in both cells,suggesting that DNMTs in mice would participate in down regulating the expression of AIRE.Conclusion:1.Estrogen can down regulate the expression of AIRE in peripheral lymph node stromal cells and splenic DCs.2.Estrogen promotes the methylation of AIRE gene and down regulates the expression of peripheral AIRE through ER-DNMTs pathway.3.It was verified that estrogen down regulated the expression of peripheral AIRE through ER-DNMTs pathway in vivo.This study confirmed that estrogen can down regulate the expression of peripheral AIRE,which may promote DNMTs expression through ERα,and mediate methylation of AIRE gene.This study provides an experimental basis for understanding the regulatory mechanism of peripheral AIRE expression,provides a possible explanation for women’s autoimmune susceptibility,and also provides a new idea for the treatment of autoimmune diseases.